Team:Heidelberg/Delftibactin/DelRest

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                                   <h1>Week 11</h1>
                                   <h1>Week 11</h1>
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During the past week we managed to amplify 11 kb as a single fragment including the genes DelA to DelF from genomic DNA from <i>D. Acidovorans</i>. Furthermore we were able to equip our biobrick backbone pSB4K5 with the desired overlaps to the genes of the Delftibactin cluster. We also succeeded in the amplification of the very last gene in our construct, DelL. However still a lot of work has to be done, since the amplification of DelF-G and DelO-P turned out to be more complicated. Since our initial plan, to amplify DelO-P with a Gibson primer which includes an overlaps to DelL and furthermore introduces an artificial ribosom binding site, turned out to be quite ambitious, we orderd shorter versions of all our Gibson primers, used to amplify genomic DNA. Using these shorter primers which should anneal better we will try to preamplify the desired genes in order to obtain a specific template for the amplification with the needed overlaps. Furthermore we will optimize the PCR conditions for the amplification of DelF-G.
During the past week we managed to amplify 11 kb as a single fragment including the genes DelA to DelF from genomic DNA from <i>D. Acidovorans</i>. Furthermore we were able to equip our biobrick backbone pSB4K5 with the desired overlaps to the genes of the Delftibactin cluster. We also succeeded in the amplification of the very last gene in our construct, DelL. However still a lot of work has to be done, since the amplification of DelF-G and DelO-P turned out to be more complicated. Since our initial plan, to amplify DelO-P with a Gibson primer which includes an overlaps to DelL and furthermore introduces an artificial ribosom binding site, turned out to be quite ambitious, we orderd shorter versions of all our Gibson primers, used to amplify genomic DNA. Using these shorter primers which should anneal better we will try to preamplify the desired genes in order to obtain a specific template for the amplification with the needed overlaps. Furthermore we will optimize the PCR conditions for the amplification of DelF-G.
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By the end of last week we decided that the short primers we had ordered in the previous week did not lead to any advantage in amplification of the missing genes from <i>D. Acidovorans </i>. Also the amplification of DelF-G did not proceed as expected. Additionly, further analysis of the Delftibactin cluster, led to the discovery of another putative promotor in front of DelO-P. Therefore we not only decided to design new primers for the region of DelFG but also modified the entire strategy and our construct in concerns of DelOP. To ensure the expression of DelOP in our target organism <i> E.coli </i> we decided to introduce this promotor in our final vector. Logically new primers were ordered for this region but as we still want to use Gibson cloning also the very last fragment of DelA-G has now to be amplified with an overlap, matching the promotor in front of DelOP. The correlating primers can be found in our new vector map.
By the end of last week we decided that the short primers we had ordered in the previous week did not lead to any advantage in amplification of the missing genes from <i>D. Acidovorans </i>. Also the amplification of DelF-G did not proceed as expected. Additionly, further analysis of the Delftibactin cluster, led to the discovery of another putative promotor in front of DelO-P. Therefore we not only decided to design new primers for the region of DelFG but also modified the entire strategy and our construct in concerns of DelOP. To ensure the expression of DelOP in our target organism <i> E.coli </i> we decided to introduce this promotor in our final vector. Logically new primers were ordered for this region but as we still want to use Gibson cloning also the very last fragment of DelA-G has now to be amplified with an overlap, matching the promotor in front of DelOP. The correlating primers can be found in our new vector map.
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                                   <h1>Week 13</h1>
                                   <h1>Week 13</h1>
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                                   <h1>Week 14</h1>
                                   <h1>Week 14</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.</p>
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                                   <h1>Week 15</h1>
                                   <h1>Week 15</h1>
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                                   <h1>Week 16</h1>
                                   <h1>Week 16</h1>
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                                   <h1>Week 17</h1>
                                   <h1>Week 17</h1>
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                                   <h1>Week 18</h1>
                                   <h1>Week 18</h1>
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                                   <h1>Week 19</h1>
                                   <h1>Week 19</h1>
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                 <h2>Methods:</h2>
                 <h2>Methods:</h2>
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Revision as of 12:10, 2 October 2013

Del Rest. Creating a 32 kbp plasmid.

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Methods: